Functional Dissection Of A Tomato Mycorrhiza-specific Plasma Membrane H~+-ATPase During Arbuscular Mycorrhizal Symbiosis

In nature ecosystems,most land plants can form root symbiosis with beneficial microorganisms for enhancing uptake of mineral nutrients or increasing adaptation to environmental challenges.Arbuscular mycorrhizal(AM)symbiosis that is formed by the interaction between the AM fungi belonging to Glomeromycotina and the roots of more than 80%land plants is the most widespread mutualistic associations in nature.The establishment of AM symbiosis between the associated partners is based on bidirectional nutrient exchange:the AM fungi colonize root cortical cells to obtain carbohydrates provided by the plants;in return,they transfer mineral nutrients,mainly P and N,from soil to the root cortex.A typical feature of the AM association is the development of highly branched,tree-like fungal structures termed arbuscules in cortical cells,which are the major sites for nutrient exchange between the two symbionts.An increasing number of plant and fungal genes associated with transporting nutrients,such as Pi and NH4+,across the intraradical symbiotic interface have been shown to be specifically induced in the arbuscule-containing cells.Transport of nutrients from interfacial apoplast into plant cells or fungal cells was thought to depend heavily on the electrochemical proton gradient generated by the H+-ATPase located on the fungal arbuscular membrane and the plant periarbuscular membrane(PAM).Multiple genes encoding different isoforms of H+-ATPases have been identified and characterized from diverse plants,mainly in Arabidopsis and rice,with their roles being associated with many Physiologyogical processes.However,limited information of the HA genes family is available to date for tomato,a model plant species of the solanaceous family.In this study,through genome-wide hunting and expression analysis of the tomato HA gene family,a new HA member,SlHA8,that was strongly responsive to AM symbiosis was identified.Functional dissection of its roles by employing the transgenic plants with enhanced or silenced expression activity revealed that SlHA8 is essential for AM colonization,arbuscule development,as well as symbiotic phosphate(Pi)and nitrogen uptake.The main results were summarized as follows:1.Genome-wide hunting and bioinformatics analysis of the tomato HA gene family revealed a total of eight paralogous genes(SlHA1-8)encoding different HA isoforms in tomato.All these genes are interrupted by multiple introns with conserved positions.Expression analysis of these HA genes showed that SlHA1,2,and 4 were widely expressed in all tissues,while SIHA5,6,and 7 were almost only expressed in flowers.SlHA8,the transcripts of which were barely detectable under normal or nutrient-/salt-stress conditions,was strongly induced in the AM fungal-colonized roots.2.Histochemical analyses showed that a 1521-bp SlHA8 promoter was able to drive the GUS reporter expression specifically in mycorrhizal tobacco roots,and were confined to distinct cells containing arbuscules.The SlHA8 promoter could also direct the GUS reporter expression specifically in colonized cells of transgenic soybean and rice mycorrhizal roots.Promoter deletion assay revealed a 223-bp promoter fragment of SIHA8 containing a variant of AM-specific cis-element MYCS(vMYCS)sufficient to confer the AM-induced activity.3.To determine whether SlHA8 functions as a membrane-localized protein,subcellular localization analysis of SlHA8 was performed.The SlHA8-GFP fusion protein was expressed under the control of either the 35S cauliflower mosaic virus promoter(35S::SlHA8-GFP)or its native promoter(ProHA8::SlHA8-GFP)in the N.benthamiana epidermal cells or soybean hairy roots.It was shown that the SlHA8-GFP fusion protein expressed in the N.benthamiana epidermal cells was exclusively localized to the plasma membrane,while in the soybean hairy roots colonized by R.irregularis,the SlHA8-GFP fusion protein showed a specific localization in the arbuscule-containing cells.These results indicate that the H+-ATPase encoded by SIHA8 is able to localized to the periarbuscular membranes upon AM symbiosis.4.Loss function of SIHA8 led to reduced colonization level and P and N uptake,truncated arbuscule morphology,and decreased H+-ATPase activity and acidification of apoplastic spaces in arbuscule-containing cells.The defective AM phenotype of slha8 could not be restored under low-N supply conditions.5.To assess whether enhancing the expression of SlHA8 may facilitate the uptake of N and P,we further generated transgenic plants overexpressing SlHA8 by using the CaMV 35S promoter.Constitutive overexpression of SIHA8 in tomato increased both P and N accumulation in the plants grown in both hydroponic solution and sand-based pot culture inoculation with AM fungi,and increased total root colonization level,but did not affect arbuscule morphology.6.Heterogenous expression of SIHA8 in rice wild-type plants promoted both P and N uptake,but had no effect on plant growth.Expression of the SIHA8 in the rice oshal mutant could fully complement its defects in arbuscule development and mycorrhizal P uptake.In conclusion,in the present study,we combined reverse genetic approaches and a cross-species complementation test to characterize the function of the tomato AM-specific H+-ATPase gene,namely,SIHA8,in AM symbiosis.A variant of the AM-responsive MYCS element that is essential for the AM-mediated regulation of dicot PT genes,was identified from its promoter,and was proven to be a mandatory requirement for conferring its AM-specific expression.We also demonstrated that SIHA 8 is not only required for AM symbiosis and mycorrhizal P and N transport in tomato,but also is able to complement the defective AM phenotype of the rice osha1 mutant.These results highlight the evolutionary conservation of the AM-specific H+-ATPase orthologs in maintaining AM symbiosis and functionality across different eudicot and monocot mycorrhizal plants.Moreover,Our results provide a reliable theory for further analysis of the function and regulation mechanism of plasma membrane H+-ATPase in AM symbiosis,and provide a new direction for plant breeding with the goal to improve crop plant productivity and plant nutrient uptake efficiency through mycorrhiza pathway.